UJMR, Volume 6 Number 2, December, 2021, pp 19 - 23 ISSN: 2616 - 0668

https://doi.org/10.47430/ujmr.2162.003

Received: 7th August, 2021 Accepted: 15th September, 2021

Comparative Studies of the Methods of Detecting Rifampicin Resistant

Mycobacterium tuberculosis among Tuberculosis Patients Attending Aminu Kano
Teaching Hospital, Kano
1
Umar, A., 1Binta, M.A., *2Hamza, J.A. , 3Mujahid, H. , and 4Igba, P.
1
Department of Microbiology, Bayero University Kano
2
Department of Pharmaceutical Microbiology and Biotechnology, Gombe State University
3
Department of Microbiology, Umaru Musa Yar’adua University, Katsina, Nigeria
4
Department of Pharmaceutical Microbiology, Ahmadu Bello University Zaria
*Correspondence to: Hamza Adamu Jabir, Department Pharmaceutical Microbiology and Biotechnology,
Gombe State University.
Email: jabirgsu@gsu.edu.ng +2347039306208
Abstract
This study was conducted to compare the specificity and sensitivity of GeneXpert MTB/Rif and
Lowenstein-Jensen (LJ) Proportion methods of detecting rifampicin-resistant Mycobacterium
tuberculosis among Acid Fast Bacilli (AFB) positive patients attending the Directly Observed
Treatment (DOT) Centre of Aminu Kano Teaching Hospital Kano. A total of 150 AFB positive
samples were collected and processed according to the guideline given by National TB and
Leprosy Control Program (2015) and WHO (2012), The result revealed that rifampicin-resistant
Mycobacterium tuberculosis (RR-TB) from the samples was very high; 66.7% and 60.8% for
GeneXpert MTB/Rif and Lowenstein-Jensen (LJ) Proportion methods respectively. Cohen’s Kappa
(interrater reliability) statistical analysis indicated a substantial agreement between GeneXpert
and LJ Proportion specificity and sensitivity (Kappa value = 0.73).
Keywords: Mycobacterium tuberculosis, GeneXpert MTB/Rif, Lowenstein-Jensen (LJ) Proportion,
Rifampicin resistance.

INTRODUCTION used in developing countries and is a proportional

Tuberculosis (TB) is an infectious disease caused
by the bacterium Mycobacterium tuberculosis
(MTB). Tuberculosis generally affects the lungs,
but can also affect other parts of the body. Most
infections do not have symptoms, known as
latent tuberculosis. About 10% of latent
infections progress to active disease which, if left
untreated, kills about half of those infected. The
classic symptoms of active TB are a chronic cough
with blood, fever, night sweats, and weight loss
(WHO, 2016).
There exist some test methods used in the
detection of resistance in TB, among which WHO
endorsed two; GeneXpert MTB/Rif assay and
Lowenstein Jensen (LJ) Proportion Method. The
GeneXpert MTB/Rif is an assay that detects the
presence of TB and also Rifampicin resistant
strain simultaneously. The phenotypic Lowenstein
Jensen (LJ) Proportion Method (LJPM) is widely
method that uses fixed concentrations of first-
line TB drugs: isoniazid (INH), rifampicin (RIF),
ethambutol (EMB), and streptomycin (SM). This
method is simple and cost-effective; however,
the results require a long time to obtain and can
be unreliable in some diagnostic examination
settings (Agonafir et al., 2010). The former
method is reported in a research conducted in
India to have 100% specificity in a large clinical
GeneXpert validation study even though the
specificity drops to 98.3% in a subsequent
multicenter study (Van et al., 2011). The use of
the later method (LJPM) is largely embraced in
our community. This study, therefore, evaluates
the specificity and sensitivity of GeneXpert
MTB/Rif assay and Lowenstein Jensen (LJ)
Proportion Method (LJPM) in the detection of RR-
TB.

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 UJMR, Volume 6 Number 2, December, 2021, pp 19 - 23
MATERIALS AND METHODS
Sample Collection and Processing:
One hundred and fifty (150) screened and
confirmed sputum samples from AFB positive
patients attending Directly Observed Treatment
(DOT) Centre of Aminu Kano Teaching hospital
Kano, were collected and processed as described
by the National TB and Leprosy Control program
SOP NTBLCP (2015) and WHO (2012); two spots of
sputum samples were collected from each
patient at least one hour apart in a sterile leak-
proof (50ml) wide mouth, screw-capped, leak-
proof falcon tube container. The patients were
instructed to rinse their mouth with water and
take 3 to 4 deep breaths, holding the breath for
3-5 seconds after each inhalation and then cough
after the last inhalation, emptying the sputum
into the falcon tube with care not to contaminate
the outside of the tube. The falcon tube screw
cap was then closed tightly and wiped with
cotton wool soaked in tuberculocidal disinfectant
(Lysol). 5 ml of the specimen was collected. The
sputum specimens were collected in an open and
well-ventilated space, the positive AFB samples
were confirmed by microscopy and then later
stored at a temperature of 8oC for further
laboratory analysis which involves genotypic
detection of Mycobacterium tuberculosis using
GeneXpert MTB/Rif assay and phenotypic
detection using culture (LJPM) method.
a. Genotypic Detection of Mycobacterium
tuberculosis Using GeneXpert Assay
GeneXpert MTB/Rif (Cepheids, USA) was used to
detect the presence of Mycobacterium
tuberculosis and also Rifampicin-resistance, the
procedure involves disinfection of the working
area then the GeneXpert MTB/Rif cartridge was
labelled with the sample number. The lid of the
sputum specimen container was unscrewed and 2
volumes of sample reagent (SR) were added
directly into 1 volume of sputum (ratio 2:1) and
the lids were closed. It was then vortex and
incubated at room temperature for 10 minutes.
After 10 minutes of incubation, it was vortex
again several times and incubated for 5 minutes.
After an additional 5 minutes of incubation, the
sample was perfectly fluids before being tested,
with no visible clumps of sputum. But if the
sample is still viscous, then another 5-10 minutes
was added before inoculating into the cartridge.
To inoculate the cartridge, a sterile pipette was
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UMYU Journal of Microbiology Research
ISSN: 2616 - 0668
used to transfer 2 ml of the liquefied sample into
an open port of the GeneXpert MTB/Rif cartridge
and closed immediately. The pipette was
discarded into a tuberculocidal disinfectant
solution for decontamination (NTBLCP, 2015).
Running of the Test
The tab, “Create a test” was clicked on a
computer. A window requesting to scan the
cartridge barcode appeared on the computer
screen, a barcode scanner was used to scan the
cartridge barcode and a window that requested
patient’s Names and Laboratory Serial numbers
was opened and filled. After this was done, the
“start” icon was clicked and so the assigned
module, as indicated by the blinking of green
light. The cartridge bay door of the selected
module was opened and the cartridge was loaded
carefully. By closing the cartridge bay door, it
automatically started running the test (NTBLCP,
2015).
b. Phenotypic Detection of
Mycobacterium tuberculosis using
Culture
The collected sputum samples were processed
before inoculation on growth media which
involves three steps; digestion (to loosen mucoid
material in sputum samples), decontamination
(to limit contamination of cultures by fast-
growing non-mycobacterial commensal
organisms), and concentration (to increase the
sensitivity of culture) (NTBLCP, 2011).
Preparation of Media and Reagent
The Lowenstein-Jensen (LJ) medium was used to
isolate Mycobacteria; and was prepared as
follows:
Preparation of Homogenized eggs
Eggs were prepared and homogenized for
incorporation into LJ medium as described by
NTBLCP SOP (2011) and WHO (2011). About 100
ml of liquid soap solution was mixed with 8 litres
of water to get an alkaline solution. One day
fresh eggs were placed in the alkaline soap
solution for 30minutes. The eggs were scrubbed
thoroughly and rinsed under running tap water.
They were then soaked in 70% alcohol for 15
minutes and placed on sterile dishes to air dry
the excess ethanol. The eggs were then cracked
carefully into a sterile measuring cylinder until
desired quantity was attained, homogenized
using a sterile blender, and filtered using sterile
gauze.
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 UJMR, Volume 6 Number 2, December, 2021, pp 19 - 23 ISSN: 2616 - 0668

Preparation and Constitution of LJ medium
The Lowenstein Jensen (LJ) medium was
prepared according to the manufacturer’s
recommendations. Exactly 37.2 g of the powder
was suspended in 600 ml of purified water
containing 12 ml glycerol. The suspensions were
mixed thoroughly, heated with frequent
agitation, and boiled for 1 minute to completely
dissolve the powder. It was then autoclaved at
121oC for 15 minutes and cooled to 45oC - 60oC.
One litre of the freshly homogenized egg was
added to the autoclaved mixture, mixed well and
dispensed into sterile tubes, and allowed to
coagulate in a slanting position at 85oC for 45
minutes in an inspissator.
Isolation
The decontaminated-digested sediments of these
sputum samples were then inoculated into the
prepared LJ media in duplicates for each sample
and then incubated aerobically at 37oC for 8
weeks. The tubes were observed daily for the
first week of incubation and weekly thereafter
till eight weeks. Caps were opened once a week
for a short interval to aerate the cultures and to
examine tubes for positive growth cultures
showing evidence of growth at any time during
this period for typical non pigmented, rough, dry
colonies on Lowenstein-Jensen medium, coupled
with the AFB positive microscopy result,
suggestive of Mycobacterium
tuberculosis complex (NTBLCP, 2011 and Gambo
et al., 2013).
Drug Susceptibility Testing using Lowenstein
Jensen (LJ) Proportion Method
Susceptibility testing of the MTBC isolates to
Rifampicin (40 μg/ml) was determined by the
proportion method using Lowenstein Jensen egg-
based slopes as described by NTBLCP (2011). For
each sample 10-1 of the MTBC was inoculated on
the drug-containing medium of the tubes. Three
drug-free LJ slopes were inoculated with 10-1, 10-
2
, 10-3 diluted suspensions of a 1.0 McFarland
standardized Inoculum. Furthermore, the
rifampicin-susceptible MTB reference strain ATCC
27294 (H37Rv) was used as a susceptible control
and known resistant strains (ATCC35838 H37Rv
for RMP) was used as resistant controls. The
slopes were incubated at 37°C and read after 4
and 6 weeks.
Reading of Results
After 28 days of incubation, slopes were observed
for growth. The average number of colonies
obtained from drug-containing slopes indicated
the number of resistant bacilli contained in the
inoculum. Dividing the number of colonies in the
drug-containing slopes by those in the drug-free
slopes gave the proportion of resistant bacilli. An
isolate was considered resistant if the proportion
of bacilli resistant to the critical concentration of
the drug exceeded 1%.
RESULTS
The result from the Genotypic detection of
Mycobacterium tuberculosis showed that out of
the AFB positive samples earlier confirmed by
microscopy, Mycobacterium tuberculosis was
detected in all the samples (100%), culture
revealed that 120 (80%) were positive as shown in
Table 1.

Table 1: Detection of Mycobacterium tuberculosis by Genotypic and Cultural Methods

Type of Assay No. Positive for M. tuberculosis Percentage (%)
Genotypic 150 100
Cultural 120 80

Table 2 shows the susceptibility of M. were resistant to rifampicin, while 73 (60.8%) out
tuberculosis to Rifampicin. Out of 150 M. of the 120 isolates detected by culture were
tuberculosis detected genotypically, 100 (66.7%) found to be rifampicin resistant.

Table 2: Susceptibility to Rifampicin Based on Cultural and Genotypic Methods

Method No. Screened No. Sensitive (%) No. Resistant (%)
Phenotypic (LJPM) 120 47 (39.2) 73 (60.8)
Genotypic (GeneXpert) 150 50 (33.3) 100 (66.7)

The specificity and sensitivity of GeneXpert MTB/Rif (genotypic) and Cultural methods of detecting
rifampicin resistance by Kappa analysis TB are presented in Table 3. The result indicated a substantial
agreement between GeneXpert and culture.

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 UJMR, Volume 6 Number 2, December, 2021, pp 19 - 23
Table 3: Comparison of Specificity and Sensitivity of Genotypic and Culture Methods
Culture Gene Xpert
Rif Resistant
Resistant 71
Sensitive 13
Total 84
Kappa value = 0.73
Interpretation:
K = 0.73 (0.61 to 0.81) indicated a substantial agreement between GeneXpert and culture results.
DISCUSSION
This study reported 100% conformity of the
GeneXpert MTB/Rif TB detection method with
that of the microscopy, as all (100%) of the AFB
positive microscopy samples were also confirmed
positive by the GeneXpert MTB/Rif genotypic
method, hence evidencing the reliability and
efficiency of both detection methods. Even
though, the microscopy detection method had
been reported to be far from being sensitive
(Peter, 2018).
With regards to the rifampicin resistance
determination, this study revealed that 66.7%
and 60.8% of the isolates were resistant to
rifampicin genotypically (GeneXpert MTB/Rif) and
phenotypically (Lowenstein Jensen (LJ)
Proportion) respectively. The rifampicin
resistance prevalence documented in this work
was higher as compared to the findings of
Omisore et al., (2018) and Peter (2018) who
respectively reported the prevalence of 10% (in
Lahore, Pakistan) and 14.7% (in the Bayelsa state
of Nigeria) using the GeneXpert MTB/Rif. The
differences observed may be due to variation in
the sampling locations and other antibiotic
resistance determining factors.
Moreso, the slight differences (of 5.9%) in the
resistance prevalence of both methods suggest
their reliabilities for resistance detection in MBT
isolates, this conforms with the findings of Munir
et al., (2018) who made a comparison of Gene
Xpert MTB/RIF Assays with Conventional Standard
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UMYU Journal of Microbiology Research
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